The classical estrogen receptor, ER?, is a ligand-regulated transcription factor important for normal development, homeostasis and reproduction, and consequently, understanding normal and pharmacological regulation of this receptor's activity is of major significance to human health. It is well accepted that the ability of ER? to regulate gene expression is dependent upon the transcriptional coregulators that ligand-bound receptors recruit to their target genes. The silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) coregulator was initially characterized as a corepressor for ER? bound to the selective estrogen receptor modulator (SERM), 4-hydroxytamoxifen (4HT), however, it can stimulate the activity of ER? bound to estradiol (E2) and this established SMRT as a dual coactivator/corepressor for ER?. Recent work demonstrates that the nature of SMRT interactions with ER? depend upon whether the receptor is bound to agonist or antagonist. Moreover, in addition to the well described activation of histone deacetylase 3 (HDAC3) by SMRT, this coregulator can also bind to a histone demethylase that promotes the transcriptional activity of ER?. The overall goal of the proposed work is to define the ability of two SMRT interacting proteins, with activating and repressive activities, respectively, to regulate ER?-dependent gene expression. The planned experiments will use state-of-the-art biological, cistromic and transcriptomic analysis to test whether differences in the nature of SMRT-ER? interactions contributes to the ability of these distinct complexes to bind and/or regulate ER? in a ligand- and gene-dependent manner, and whether the composition or activity of the repressive complexes can be regulated by cellular pathways. Using novel 3-dimensional culture of mammary epithelial cells that maintain steroid responsiveness and mouse models that enable the precise evaluation of the HDAC3 activation activity of SMRT in vivo, the ability of SMRT to regulate ER? transcriptional activity as well as mammary gland development will define the biological importance of SMRT for regulation of ER? responses to both agonists and antagonists in a physiological setting. Together, these planned studies will evaluate the hypothesis that differences in the proteins that SMRT recruits to ER? target genes are critical determinants of the effect of this coregulator on ER?-dependent gene expression. In so doing, the proposed work will provide critical insight into mechanisms of action of SMRT independent of HDAC3, and the contribution of SMRT to ER? regulation in estrogen-dependent disease such as breast cancer.